Isolated phenylalanine dehydrogenase gene and process for production of phenylalanine dehydrogenase

ABSTRACT

An isolated gene coding for phenylalanine dehydrogenase of a microorganism belonging to a genus selected from the group consisting of the genera Bacillus and Sporosarcina origin; plasmids containing the gene; microorganism transformed with the plasmid, a process for the production of phenylalanine dehydrogenase using the microorganism; and a process for the production of L-phenylalanine using the enzyme.

This application is a divisional of Ser. No. 07/084,238 filed Aug. 11,1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to genes coding for phenylalaninedehydrogenase, plasmids containing the gene, microorganisms transformedwith the plasmid, a process for the production of phenylalaninedehydrogenase, and a process for the production of L-phenylalanine usingan enzyme.

2. Description of the Related Art

Attempts have been made to produce L-amino acids using α-ketocarboxylicacid as a substrate. For example, a process for preparing L-glutamicacid by adding α-ketoglutarate and various kinds of amino acids tomicrobial cells (Katagiri et. al. Amino Acid and Nucleic Acid, 2, 18,1960); a process for obtaining L-phenylalanine by adding L-glutamic acidor L-aspartic acid to a reaction mixture containing phenylpyruvic acid(Asai et. al., Amino Acid and Nucleic Acid, 2, 114 (1960); and a processfor synthesizing L-tryptophan by adding L-glutamic acid or L-asparticacid to a reaction mixture containing indolepyruvic acid (Aida et. al.,Journal of General and Applied Microbiology, 4, 200, 1958, have beendisclosed.

Japanese Unexamined Patent Publication No. 60-164493 describes a processfor the production of L-phenylalanine by either culturing one of variouskinds of microorganisms with phenylpyruvic acid and an amino groupdonor, or incubating cells of that microorganism or a treated product ofthe cells with phenylpyruvic acid and an amino group donor. However,this specification does not disclose just what kind of enzymesparticipate in the reaction. Moreover, such processes employ aminoacids, which are an expensive amino group donor.

All of the above-mentioned processes use, as an amino group donor of anaimed amino acid, another amino acid, and are fundamentally differentfrom a process of the present invention which uses an ammonium ion as anamino group donor, which is not so expensive. Namely, the prior artprocesses are more expensive than the present process. Moreover, theenzymes involved in the prior art process are different from those ofthe present process.

Japanese Unexamined Patent Publication No. 60-43391 discloses a processfor production of L-amino acid wherein a microorganism capable ofconverting an α-keto acid to a corresponding L-amino acid is cultured,and during the culturing, the α-keto acid is fed into the culturingmedium to convert the α-keto acid to the L-amino acid. According to thereaction mechanism suggested in the specification, as an amino groupdonor for the formation of an aimed L-amino acid from a correspondingα-keto acid, L-glutamate is used, which means that the reaction iscarried out by an amino transferase. Moreover, the application disclosesonly Brevibacterium, Corynebacterium, and Escherichia coli asmicroorganisms involved.

Japanese Unexamined Patent Publication No. 59-198972 describesphenylalanine dehydrogenase and a process for the production ofL-α-amino carboxylic acid using that enzyme. However, the phenylalaninedehydrogenase described therein is derived from Brevibacterium, and thespecification does not suggest that Sporosarcina and Bacillus produce asimilar enzyme. Moreover, the disclosed phenylalanine dehydrogenase hasa molecular weight of 130,000±10,000 and consists of subunits having amolecular weight of 66,000±5,000 and, therefore, is different from thepresent phenylalanine dehydrogenase.

Japanese Unexamined Patent Publication No. 60-160890 discloses a processfor the production of L-phenylalanine by either culturing one of variouskinds of microorganisms with phenylpyruvate in the presence of an energysource, an inorganic ammonium compound or urea, and oxygen, or byincubating a cultured product of the microorganism or treated productthereof with phenylpyruvate in the presence of an energy source, aninorganic ammonium compound or urea, and oxygen. However, thespecification does not suggest the kind of enzymes involved in theprocess, and the process is supposed to be essentially a fermentationprocess, due to the necessity for the presence of oxygen. Moreover, thespecification does not refer to Sporosarcina.

Japanese Unexamined Patent Publication No. 60-24192 discloses theproduction of L-phenylalanine using a Corynebacterium strain transformedwith a plasmid containing genes related to L-phenylalanine productionderived from Corynebacterium.

Japanese Unexamined Patent Publication No. 60-62992 discloses thecloning of genes related to the synthesis of L-phenylalanine inEscherichia coli, and the expression of that gene under the control of atrp promoter to produce L-phenylalanine.

Japanese Unexamined Patent Publication Nos. 60-66984 and 60-210993disclose the synthesis of L-phenylalanine using Brevibacteriummicroorganism transformed with a plasmid incorporating a gene for anenzyme related to L-phenylalanine synthesis, which gene is fromL-glutamate-producing Coryneform. phenylalanine dehydrogenase genes ofBacillus and Sporosarcina origin, plasmids containing that gene,microorganisms transformed with the plasmid, and a process for theproduction of L-phenylalanine have not been described.

Note, microorganism producing phenylalanine dehydrogenase, a process forproduction of phenylalanine dehydrogenase, phenylalanine dehydrogenaseper se, and a process for production of α-amino acid includingL-phenylalanine are described in detail in E.P.C publication No. 0 206460.

Generally, the ability of wild microorganism strains to produce usefulsubstances is low, and therefore, when the production of usefulsubstances is intended, using a microorganism, an improvement ofmicroorganism is attempted to increase the ability of the microorganismto produce a target substance. However, conventional processes for theimprovement of a microorganism, such as artificial mutagenesis using,for example, a chemical mutagen or physical mutagen such as UV rays, aretime-consuming and depend greatly on chance.

SUMMARY OF THE INVENTION

Accordingly, in the present invention, a gene technology is used toenhance the ability of microorganisms to produce phenylalaninedehydrogenase.

The present invention provides an isolated gene coding for phenylalaninedehydrogenase of a microorganism belonging to the genus selected fromthe group consisting of the genera Bacillus and Sporosarcina origin.

The present invention also provides expression plasmids containing theabove-mentioned genes.

The present invention also provides microorganisms transformed with theabove plasmids.

The present invention also provides a process for the production of anphenylalanine dehydrogenase comprising the steps of:

culturing the above-mentioned microorganism, and

recovering the phenylalanine dehydrogenase from the cultured product.

The present invention further provides, a process for the production ofan L-phenylalanine comprising the steps of:

reacting phenylpyruvic acid or a salt thereof with ammonium ion in anaqueous medium in the presence of the phenylalanine dehydrogenaseproduced by the above-mentioned process and a reducing agent to formL-phenylalanine, and

recovering the L-phenylalanine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 represent a process for the construction of plasmidspBPDH3, pBPDH1-DBL, and pBPDH1-DBR from a plasmid pBPDH1, all containingan phenylalanine dehydrogenase gene;

FIGS. 3-1 to 3--3 represent a nucleotide sequence (upper line) of a DNAfragment in the plasmid pBPDH1 containing a region coding for thepresent phenylalanine dehydrogenase, and an amino acid sequence (lowerline) corresponding to the above-mentioned nucleotide sequence;

FIG. 4 represents restriction endonuclease cleavage maps for plasmidspSPDH1 and pSPDH2;

FIGS. 5-1 to 5-3 represent a nucleotide sequence (upper line) of a DNAfragment in the plasmid pSPDH1 containing a region coding for thepresent phenylalanine dehydrogenase, and an amino acid sequence (lowerline) corresponding to the above-mentioned nucleotide sequence; and

FIG. 6 represents a restriction endonuclease cleavage map for theplasmid pBBPDH19.

DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the present invention, DNA is extracted from amicroorganism belonging to the genus Bacillus or Sporosarcina andcapable of producing phenylalanine dehydrogenase, an phenylalaninedehydrogenase gene is obtained by cleaving the extracted DNA withappropriate restriction enzymes, the gene is introduced to anappropriate vector to construct an expression plasmid, and amicroorganism is transformed with the expression plasmid to form anphenylalanine dehydrogenase-producing transformant. This transformedmicroorganism is used to produce an phenylalanine dehydrogenase, whichis then used to produce L-phenylalanine.

1. Microorganism as gene source

Microorganisms which can be used as a gene source for the presentinvention are those belonging to the genus Sporosarcina or Bacillus andcapable of producing phenylalanine dehydrogenase. Such micro-organismsmay be selected from deposition libraries or may be isolated fromnatural sources.

The microorganisms belonging to the genus Sporosarcina includeSporosarcina ureae. Such species include, for example, strainsSporosarcina ureae IFO 12698 and Sporosarcina ureae IFO 12699 (ATCC6473) as species selected from deposition library, and Sporosarcinaureae SCRC-R04 first isolated by the present inventor. The formerstrains can be obtained from the Institute for Fermentation Osaka (IFO),17-85 Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan, or theAmerican Type Culture Collection (ATCC), 12301, Parklawn Drive,Rockville, Md. 20852, U.S.A.; and the latter strain, Sporosarcina ureaeSCRC-R04, was deposited with the Fermentation Research Institute Agencyof Industrial Science and Technology, Ministry of International Tradeand Industry (FRI), 1-1-3 Yatabe-cho Higashi, Tsukuba-gun, Ibaraki-ken,Japan, as FERM P-8178, on Apr. 16, 1985, and transferred to theinternational deposition under the Budapest Treaty on the InternationalRecognition of the Deposit of Micro-organisms for the Purposes of PatentProcedure (Budapest Treaty) as FERM BP-1012 on Apr. 3, 1986.

The microorganisms which can be used as a gene source in the presentinvention belonging to genus Bacillus include, for example, Bacillusalvei IFO 3343; Bacillus thiaminolyticus IAM 1034 deposited with FRI asFERM P-8528 on Nov. 28, 1986; Bacillus badius IAM 11059, ATCC 14574,deposited with FRI as FERM P-8529 on Nov. 28, 1985; Bacillus sphaericusIFO 12622; Bacillus sphaericus IAM 1228, deposited with FRI as FERMP-8527 on Nov. 28, 1985. All of the above-mentioned strains are listedin catalogs published by IFO, ATCC, or for IAM by the JapaneseFederation of Culture Collections of Microorganisms (JFCC), Institute ofMedical Science, University of Tokyo, Shiroganedai 4-6-1, Minato-ku,Tokyo 108, Japan, and are available to the public.

Moreover, the microorganism which can be used as a gene source in thepresent invention includes new strains isolated by present inventors,such as Bacillus sp. SCRC-R53b; Bacillus sp. SCRC-R79a deposited withFRI as FERM P-8179 on Apr. 16, 1985, and transferred to theinternational deposition under the Budapest Treaty as FERM BP-1013 onApr. 3, 1986; Bacillus sp. SCRC-101A; and Bacillus sp. SCRC 114Ddeposited with FRI as the international deposition FERM BP-1011 on Apr.3, 1986.

According to Bergey's Manual of Determinative Bacteriology, the eighthedition, 1974, the above-listed strains were identified as follows:

(a) SCRC-R04 is aerobic, motile, capable of forming spores, Grampositive, and 2 to 4 cocci. Therefore the strain belongs to genusSporosarcina. Since Sporosarcina includes Sporosarcina ureae as solespecies, and the above-mentioned properties of SCRC-R04 are almostconsistent with those described in the literature, the strain SCRC-R04is identified to be Sporosarcina ureae.

(b) All SCRC-R53b, SCRC-R79a, SCRC-101A and SCRC-114D are Gram positive,rod, capable of forming endospores and forming catalase. Therefore, theybelong to Bacillus.

Details of the properties of the above-mentioned strains are describedin E.P.C. patent publication No. 0 206 460.

2. Cloning of phenylalanine dehydrogenase gene and construction ofexpression plasmid

According to the present invention, any of the above-mentionedphenylalanine dehydrogenase-producing microorganisms can be used as thegene source, and an embodiments wherein Bacillus sphaericus SCRC-R79a(FERM BP-1013) Sporosarcina ureae SCRC-R04 (FERM BP-1012) and Bacillusbadius IAM 11059(FERM P-8529) are used is definitively described herein.The process for gene cloning is described in detail in the Examples.

Although as an expression control region for the present phenylalaninedehydrogenase gene that naturally accompanies the phenylalaninedehydrogenase gene can be used, to enhance the expression or to makepossible the induction of the expression, a foreign promoter/operator ispreferably used. If E. coli is used as a host, trp, tac, lacUV5, P_(L),P_(R), 1_(PP) or the like may be used as a promoter/operator, and as theSD sequence, that of a trp leader peptide, lacZ, metapyrocatechase gene,cII gene or the like may be used. Moreover, a transcription terminator,such as a rrnBT₁, T₂ terminator of an E. coli ribosome gene can beprovided downstream of the coding region. Moreover, for the productionof an phenylalanine dehydrogenase gene, a host/vector system ofSaccharomyces cerevisiae can be used. As the yeast promoter, a promoterof an alcohol dehydrogenase gene, a promoter of an acidic phosphatasegene, a promoter of a glyceraldehyde-3-phosphate dehydrogenase, apromoter of an enolase, or the like, can be used. The yeast plasmidpreferably contains a yeast replication origin and a selective maker fora selection of yeast cells containing the plasmid, for example, a geneproviding prototrophy to a auxotrophic host such as an LEU, TRP, HISgene or the like.

Generally, an amount of the expression of a particular protein in yeastcells depends on the copy number of the gene to be expressed, theefficiency of the transcription, the stability of the mRNA, theefficiency of the translation, and the stability of the producedprotein. To modify expression control regions such as the promoter, SDsequence, terminator and the like, preferably the plasmid is small.Note, the smaller the plasmid, the greater the increase in the copynumber. Therefore, after a DNA fragment containing an phenylalaninedehydrogenase gene is inserted into a plasmid, preferably the subcloningof the plasmid is repeated to eliminate a non-coding region of the DNAfragment and thus reduce the size of the plasmid.

According to the present invention, preferably the host is an E. colistrain, for example, a strain derived from E. coli K-12, such as JM83,JM101, JM103, JM105, JM109, RR1, RB791, W3110, C600, HB101, DH1 and thelike. On the other hand, as the yeast host, preferably Saccharomycescerevisiae strains such as AH22, DC5, D-13-1A, YNN140 and the like areused.

3. Production of phenylalanine dehydrogenase

According to the present invention, phenylalanine dehydrogenase can beproduced by a conventional method used for the production of a proteinby genetic engineering procedures. For example, a microorganism such asE. coli, transformed with an expression plasmid containing phenylalaninedehydrogenase gene, is cultured in an appropriate medium, and when thecell concentration reaches a predetermined level, an inductiontreatment, which depends on nature of the control region of the plasmidused, is carried out to produce an phenylalanine dehydrogenase.

L-phenylalanine is recovered and purified from the cultured brothaccording to a combination of conventional procedures used for thepurification of an enzyme. For example, a cultured broth is centrifugedto collect bacterial cells. The cells are then disrupted by aconventional method such as ultrasonication or Dynomill treatment, anddebris eliminated by a conventional method such as centrifugation orfiltration to obtain a supernatant or filtrate containing the aimedenzyme. Further purification, for example, protamine sulfate treatment,streptomycin sulfate treatment, salting out, organic solventprecipitation, absorption chromatography, ion exchange chromatography,gel filtration chromatography, and crystallization using, for example,ammonium sulfate or polyethyleneglycol, may be used.

The activity of the present phenylalanine dehydrogenase is determinedaccording to the following procedures: 100 μ moles of glycine-KCl-KOHbuffer (pH 10.5), 2.5 μ moles of AND⁺, 10 μ moles of L-phenylalanine,and an appropriate amount of sample are mixed to a total volume of 1 mlto react the components, and the increase of an amount of NADH ismeasured according to the increase of absorption at 340 nm. An amount ofthe enzyme which increases an amount of NADH by 1 μ mole per 1 minute isdefined at 1 unit.

4. Production of L-phenylalanine

According to one embodiment of the present process, a phenylpyruvicacid, NADH and ammonium ion are reacted under the presence ofphenylalanine dehydrogenase to form L-phenylalanine, and theL-phenylalanine is recovered.

The forms of the enzyme preparations of phenylalanine dehydrogenase arenot limited. The preparations include, for example, a completelypurified enzyme; cultured broth containing cells; living cells; driedcell powders prepared by treating cells with, for example, acetone orethanol; disrupted cells; and partially purified enzyme preparationspurified to various purification stages. Moreover, immobilized enzymepreparations, such as immobilized enzyme and immobilizedenzyme-containing products prepared according to conventional proceduresmay be used. For industrial production, living cells or immobilizedenzyme preparations are preferably used.

An amount in a reaction medium of phenylalanine dehydrogenase derivedfrom the above-mentioned enzyme preparation is not critical, butpreferably is within about 10 to 10,000 units per 1 liter, depending onthe nature and amount of the substrate α-ketocarboxylic acid and otherconditions.

As a substrate, both phenylpyruvic acid and its salt may be used. Thesalts include, for example, sodium salt, potassium salt, lithium salt,and calcium salt, etc. An amount of phenylpyruvic acid or salt thereofin the reaction medium is not critical, but is preferably about 1 to 500g/l depending on the concentration of the enzyme. When the substrate isused in a lower concentration, it can be used as free acid, and whenused in a relatively high concentration, it is preferably used as a saltto simplify the adjustment of the pH of a reaction medium. The sodiumsalt of phenylpyruvic acids in a high concentration is not dissolved,but the presence of a solid salt in the reaction medium is notdisadvantageous. When an ammonium salt of phenylpyruvic acid is used,the ammonium salt may act as a source of ammonium ion as well as asource of phenylpyruvic acid. In the batch-wise reaction, phenylpyruvicacid or salt thereof may be added to the reaction medium at one time atthe start of the reaction, or added in portions or continuously duringthe reaction. The salts of phenylpyruvic acid may be those commerciallyavailable or those prepared by neutralization of phenylpyruvic acid witha corresponding base, such as sodium hydroxide or ammonia.

As a source of ammonium ion, an ammonium salt, such as ammonium chlorideor ammonium sulfate may be used. Moreover, ammonia gas or a aqueousammonium hydroxide may be introduced in the reaction medium to maintainthe pH value within a predetermined range. As described above, when anammonium salt of phenylpyruvate acid is used as a substrate, the saltalso serves as a source of ammonium ion. The amount of ammonium ion usedis stoichiometric or more, in relation to the mol amount ofphenylpyruvic acid used, and more specifically, about 1 to 100 molamount in relation to the mol amount of phenylpyruvic acid used. Byincreasing the amount of ammonium salt used, the equilibrium of theenzyme reaction involved is forced to the side of L-phenylalanineformation, resulting in an increase in the yield of L-phenylalanine inrelation to phenylpyruvic acid.

NADH may be used in an equivalent amount with phenylpyruvic acid. SinceNADH is very expensive, however, from the industrial point of view, inaddition to the reaction system wherein phenylpyruvic acid isreductively aminated with NH₄ ⁺ and NADH to form L-phenylalanine andNAD⁺, an NADH regenerating system wherein the formed NAD⁺ is re-reducedto NADH is preferably used. As such an NADH regenerating system, acombination of an enzyme which converts NAD⁺ to NADH and a substrate forthe reaction, for example, a combination of formate dehydrogenase (EC1.2.1.2) and formate, L-glutamate dehydrogenase (EC 1.4.1.2) andL-glutamate, alcohol dehydrogenase (EC 1.1.1.1) and ethanol, aldehydedehydrogenase (EC 1.2.1.3) and acetaldehyde, or a combination ofglucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glucose-6-phosphatemay be used. Moreover, the reduction of NAD⁺ to NADH with hydrogenase(EC 1.18.3.1) using molecular hydrogen as an electron donor or thereduction of NAD⁺ to NADH accompanied by the oxidation of methylbiologenor dihydrolipoamide with diaphorase (EC 1.6.4.3) may be used. Whereformate dehydrogenase and formate are used, simultaneously with thereduction of NAD⁺ to NADH, formic acid is oxidized to carbon dioxidegas, which is easily eliminated from the reaction system, with theresult that the equilibrium of the reaction is forced in the desireddirection. Therefore, as the NADH regenerating system, the combinationof formate dehydrogenase and formate is especially preferable.

Formate dehydrogenase is commercially available, or prepared fromCandida boidinii No. 2201 (AKU 4705) or Hansenula polymorpha ATCC 26012according to a known procedure described by Kato et al., Agriculturaland Biological Chemistry, 38, 111-116 (1974). Where formatedehydrogenase is used in the form of cells containing the same, thetreatment of the cells can be carried out according to a known proceduredescribed by Izumi et al. in Journal of Fermentation Technology, 61,135-142 (1983).

The concentration of the enzyme for the NADH regenerating system varies,depending on the concentration of the phenylalanine dehydrogenase, etc.,and is generally a concentration at which NAD⁺ is reduced to NADH at arate corresponding to a rate at which an α-ketocarboxylic acid isreductively aminated, i.e., NAD⁺ is formed. For example, where formatedehydrogenase is used as an enzyme for the NADH regenerating system incombination with 10 to 10,000 units/l of phenylalanine dehydrogenase,the concentration of the formate dehydrogenase is preferably about 10 to10,000 units/l. As a substrate for formate dehydrogenase, a salt offormic acid, such as sodium formate, potassium formate or ammoniumformate is conveniently used. The amount of formate is preferably a oneto two equivalent amount of the α-ketocarboxylate used. Where the NADHregenerating system is used, NAD⁺ or NADH may be added to 0.1 to 10 mM,which is a usual physiological concentration.

As a reaction medium water or various kinds of an aqueous solution, forexample, an aqueous buffer solution, or an aqueous solution containingan organic solvent such as acetone, acetonitrile, dimethylsulfoxide ordimethylformamide may be used. The buffer solutions include Tris-HClbuffer, glycine-NaOH buffer, etc.

Where the NADH regenerating system is not used, reaction is carried outat a pH suitable for the reductive amination of phenylpyruvic acid bythe phenylalanine dehydrogenase used. Phenylalanine dehydrogenasederived from Sporosarcina is used at a pH of 8 to 10, preferablyapproximately at a pH of 9, while phenylalanine dehydrogenase derivedfrom Bacillus is used at a pH of 9 to 11, preferably approximately at apH of 10. Where the NADH regenerating system is used in combination withthe reductive amination of phenylpyruvic acid, the pH value of thereaction medium is selected as a value within the range wherein both thereductive amination of α-ketocarboxylic acid and the reduction of NAD⁺to NADH proceed satisfactorily. Such a pH range is that where acombination of phenylalanine dehydrogenase from Sporosarcina and formatedehydrogenase from Candida boidinii is used, usually a pH of 7.5 to 9.5,preferably a pH of 8.0 to 9.0; while where a combination ofphenylalanine dehydrogenase from Bacillus and formate dehydrogenase fromCandida boidinii is used, the pH range is usually 8 to 10, preferably8.5 to 9.5.

The reaction temperature is selected under the same consideration asmade for the selection of the pH range, and the reaction temperature isusually 20° C. to 50° C., preferably 25° C. to 40° C.

The reaction term is not critical, and is selected so that a substratephenylpyruvic acid is converted to L-phenylalanine at a satisfactoryconversion ratio, depending on the concentration of the substrate andamount of enzymes present in the reaction medium.

The reaction may be carried out batch-wise or continuously.

According to another embodiment of the present process, a substratephenylpyruvic acid is converted to L-phenylalanine, in the presence of agrowing culture such as a medium containing living cells, living cellsseparated from cultured broth, cells treated to an extent wherein anenzyme system necessary for the conversion of phenylpyruvic acid toL-phenylalanine is not destroyed, and in the presence of an energysource, without the artificial addition of NADH, NAD⁺, and the NADHregeneration system. The energy source is added to a reaction medium,and the energy source may act as an electron donor for a reductiveamination of phenylpyruvic acid. The energy sources include, forexample, sugars such as arabinose, ribose, ribulose, xylose, fucose,rhamnose, fructose, galactose, gluconate, trehalose, glucose, mannitol,mannose, sorbitol, sorbose, inositol, lactose, maltose, sucrose,raffinose, glycerine, starches, inulin, glycogen,carboxymethylcellulose, and the like. Further, the energy sourcesinclude alcohols such as ethanol and methanol, organic acids such aspropionic acid, acetic acid, formic acid, citric acid, pyruvic acid,succinic acid, malic acid, α-keto-glutaric acid, and the like.

For the second embodiment, the reaction medium, reaction pH, reactiontemperature, and other conditions are selected as described for thefirst embodiment. The second embodiment also does not require theaerobic condition.

L-phenylalanine thus formed is recovered and purified according to anyconventional procedure. For example, the reaction mixture is added totrichloroacetic acid to precipitate protein, and the precipitate, ifany, as well as the cells, is eliminated by filtration or centrifugationto obtain a filtrate or supernatant containing the L-phenyl alanine. Theproduct in the filtrate or supernatant is then purified by, for example,ion exchange resin, and finally crystallized.

A quantitative analysis of L-phenylalanine is carried out by bioassayusing, for example, Leuconostoc mesenteroides ATCC 8042, or by paperchromatography wherein a sample containing L-amino acid is separated infilter paper, a spot of L-phenylalanine is developed with ninhydrin, andthe developed spot is eluted for spectro-photometric analysis.

Examples

The present invention will now be further illustrated by, but is by nomeans limited to, the following examples.

Example 1. Preparation of chromosomal DNA containing phenylalaninedehydrogenase gene (1)

Bacillus sphaericus SCRC-R79a (FERM BP-1013) was inoculated into 3 of amedium containing 2 g/l of L-phenylalanine, 5 g/l of yeast extract, 10g/l of peptone, 2 g/l of K₂ HPO₄, 1 g/l of NaCl and 0.5 g/l of MgSO₄·7H₂ O, pH 7.0), and cultured therein for about 10 hours at 30° C., withshaking, to a cell concentration of OD₆₁₆ =1.1, which value has beenpreviously confirmed to correspond to the logarithmic growth phase.

The cultured product was centrifuged to collect cells, and 10 g of thewet cells was used to extract chromosomal DNA according to the Doimethod (see, literature 1). Namely, the cells were suspended in 40 ml ofa TEN buffer (10 mM Tris-HCl, pH 6.7, 1 mM EDTA, and 10 mM NaCl), andthe suspension was centrifuged to collect the cells. The precipitatedcells were suspended in 20 ml of a SET buffer (20% sucrose, 50 mMTris-HCl, pH 7.6, and 50 mM EDTA), 10 mg of lysozyme was added to thesuspension, and the whole was incubated at 37° C. for 30 minutes. Afterthe formation of spherophasts was confirmed under a microscope, 10 ml ofa TEN buffer and 0.25 g of sodium dodecyl sulfate was added to theabove-mentioned suspension to lyze the spherophasts. The lysate wasextracted with phenol/chloroform in a conventional manner followed by anethanol precipitation of DNA, which was then picked up with a glass bar.The DNA was dissolved in 10 ml of a TEN buffer, 0.5 mg of RNase wasadded to the solution, and the whole was incubated at 37° C. for twohours. 1 mg of pronase was added to the solution, which was thenincubated at 37° C. for one hour.

After phenol/chloroform extraction, DNA was precipitated with ethanoland dried under a reduced pressure. The dried DNA was dissolved in a TENbuffer.

Example 2. Insertion of chromosomal DNA into vector (1)

Plasmid pUC9 or pBR322 was used as a vector, and accordingly, 10 μg ofpUC9 or pBR322 was digested with a restriction endonuclease Hind III at37° C. for two hours, treated with calf thymus phosphatase at 37° C. forone hour, and then treated with phenol.

The thus-treated solution was subjected to agarose (1%) electrophoresis,and a 2.7 kb DNA fragment from the pUC9 or a 4.4 kb DNA fragment fromthe pBR322 was isolated by electroelution. The elute was extracted withphenol/chloroform followed by ethanol precipitation of a vector DNA,which was then dissolved in 20 μl of sterile water.

On the other hand, 50 μg of chromosomal DNA prepared in Example 1 wasdigested with 50 units of Hind III at 37° C. for 16 hours, treated withphenol/chloroform, and precipitated with ethanol. The precipitated DNAwas then dissolved in 50 μl of sterile water, and 0.5 μg of the vectorDNA and 2 μl of the chromosomal DNA, prepared as above, were mixed andligated using a T4 DNA ligase in the presence of ATP and dithiothreitolat 12.5° C. for 16 hours.

The reaction mixture was used to transform E. coli according to aconventional procedure. The host used was E. coli K-12/JM103 for thevector pUC9, and E. coli K-12/PR1 for the vector pBR322.

Example 3. Screening of phenylalanine dehydro genase positive clone

The transformation mixture was plated on nitrocellulose filters put onLB plate supplemented with 50 μg/ml ampicillin at a ratio of 500 to 1000clones per filter. After culturing at 37° C. for 16 hours, the culturewas replicated on fresh nitrocellulose filters, which were thenincubated at 37° C. for three hours.

The nitrocellulose filter was soaked in a lysozyme solution (5 mg/mllysozyme, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) and allowed to stand ata room temperature 10 for several tens of minutes to lyse the E. colicells. The filter was then soaked in 0.5 ml of a tetrazolium solution(0.2 M Tris-HCl, pH 8.5, 1.5 mM NAD⁺, 0.8 mM2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride and 0.32 mMphenazine methosulfate), and transformants providing a deep reddishviolet color were selected.

As a result, about 2000 each of ampicillin resistant transformants fromvectors pUC9 and pBR322 provided one phenylalanine dehydrogenasepositive clone respectively.

Example 4. Analysis of plasmid from transformant

Plasmids were extracted from phenylalanine dehydrogenase positiveclones, and a recombinant plasmid derived from pUC9 as a vector wasdesignated as pBPDH1 Each plasmid was cleaved to prepare a restrictionendonuclease cleavage map thereof. The map is set forth in FIG. 1.

A nucleotide sequence of a DNA fragment containing a region coding forphenylalanine dehydrogenase in the plasmid pBPDH1, and an amino acidsequence corresponding to the nucleotide sequence, are set forth inFIGS. 3-1 to 3--3. This sequence is also present in plasmids pBPDH1-A aspBPDH1-BS, as well as other plasmids of the present invention shown inFIGS. 1 and 2.

Example 5. Subcloning of pBPDH1(FIG. 1)

In this example, 10 μg of plasmid pBPDH1was digested with Hind III, thedigestion product was subjected to agarose gel (0.7%) electrophoresis,and a DNA fragment of about 8 kb was isolated by electroelution. Then, 2μg of pUC9 was digested with Hind III and treated with calf thymusphosphatase followed by phenol/chloroform. Both reaction mixtures weremixed, purified by Elutip-d column (Schleicher & Schuell), and the DNAwas precipitated with ethanol.

The precipitate was then dissolved in a T4 ligase buffer (50 mMTris-HCl, pH 7.4, 10 mM MgCl₂, 10 mM DTT, and 1 mM ATP), and ligationwas carried out using T4 ligase.

The reaction mixture was used to transform E. coli JM103. clone showedas a white color on an LB plate containing 50 μg/ml ampicillin, 0.3 mMisopropyl-B-D-thiogalactoside, and 0.03%5-bromo-4-chloro-3-indoryl-β-D-galactoside, which indicates a lack ofβ-galactosidase activity. A plasmid was extracted from these clones, anda recombinant plasmid wherein a Hind III fragment of about 6 kb wasinserted in the Hind III site of pUC9 was selected. The recombinantplasmid was designated as pBPDH1-A.

Example 6. Construction of plasmid pBPDH3 (FIG. 1)

In this example, 2 μg of pBR322 was digested with Hind III, treated withcalf thymus phosphatase, followed by phenol/chloroform. To the treatedproduct was added the DNA fragment of about 6 kb isolated in Example 5,and the DNA mixture was purified by Elutip-d column and precipitatedwith ethanol. The precipitate was dissolved in 10 μl of a T4 ligasebuffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂ , 10 mM DTT and 1 mM ATP),and ligation was carried using a T4 ligase. The reaction mixture wasused to transform E. coli RR1. The transformants were screened accordingto a conventional procedure, and a plasmid pBPDH3 was obtained.

Example 7. Subcloning of pBPDH1-A (FIG. 1)

In this example, 10 μg of pBPDH1-A was digested with Bam HI and Sal I,the digest was subjected to agarose gel (0.7%) electrophoresis, and aDNA fragment of about 3 kb was isolated by electroelution.

Then, 2 μg of pUC9 was digested with Bam HI and Sal I, and treated withcalf thymus phosphatase, followed by phenol/chloroform. According to thesame procedure as described in Example 5, both reaction mixtures weremixed, and after purification by Elutip-d column, DNA was ligated usinga T4 ligase, and the reaction mixture was used to transform E. coliJM103. Ampicillin resistant transformants were screened according to thesame procedure as described in Example 5 to select white clones which donot exhibit β-galactosidase activity. Plasmids were extracted from theseclones, and on the basis of restriction enzyme digestion patterns, arecombinant plasmid wherein a Bam HI - Sal I fragment of about 3.2 kbwas inserted in the Bam HI - Sal I sites of pUC9 was selected anddesignated as pBPDH1-BS. The nucleotide sequence of a DNA fragment inthe plasmid pBPDH1-BS containing a region coding for phenylalaninedehydrogenase, and a corresponding amino acid sequence, are set forth inFIGS. 3-1 to 3--3. The nucleotide sequence was determined according to adideoxy chain termination method (literature 4).

Example 8. Subcloning of pBPDH1-BS (FIG. 2)

In this example, 10 μg of pBPDH1-BS was digested with Dra I, and thedigested DNA was precipitated with ethanol. The Dra I-digested DNA and0.01 OD₂₆₀ units of a Bam HI linker phosphorylated at the 5' end thereofwere ligated in 20 μl of a T4 ligase buffer using 350 units of a T4ligase at 22° C. for 5 hours, and the ligated DNA was precipitated withethanol. The thus-prepared DNA was digested with Bam HI, the digest wassubjected to agarose gel (0.7%) electrophoresis, and a DNA fragment ofabout 2 kb was isolated by electro-elution.

On the other hand, 2 μg of pUC9 was digested with Bam HI, treated withcalf thymus phosphatase, followed by phenol/chloroform. According to thesame procedure as described in Example 5, both reaction mixtures weremixed, purified by Elutip-d column, and ligated using a T4 ligase Thereaction mixture was used to transform E. coli JM109. According to thesame procedure as described in Example 5, β-galactosidase negativeclones were selected.

Plasmids were extracted from these clones, and on the basis of therestriction enzyme digestion pattern, a recombinant plasmid wherein aDNA fragment of about 1.8 kb was inserted in the Bam HI site of pUC9 wasselected and designated as pBPDH1-Dr.

Example 9. Subcloning of pBPDH1-Dr (FIG. 2)

In this example, 10 μg of pBPDH1-Dr was digested with Bal I andprecipitated with ethanol. The Bal I-digested DNA and 0.01 OD₂₆₀ unitsof a Bam HI linker phosphorylated at the 5' end thereof were ligated in10 μl of a ligase buffer using 350 units of a T4 ligase at 16° C. for 6hours, and the ligated DNA was precipitated with ethanol. The DNA wasdigested with Bam HI, the digest was subjected to agarose gel (0.7%)electrophoresis, and a DNA fragment was isolated by electroelution. Theelute was treated three times with phenol/chloroform, and DNA wasprecipitated with ethanol.

On the other hand, 2 μg of pUC8 was digested with Bam HI, and treatedwith calf thymus phosphatase, followed by phenol/chloroform, and thedigested DNA was precipitated with ethanol.

Both the reaction mixtures, prepared as described above were ligatedusing a T4 ligase, and the reaction mixture was used to transform E.coli JM109. According to the same procedure as described in Example 5,clones which did not exhibit β-galactosidase activity were selected.Plasmids were extracted from these clones, and on the basis of therestriction endonuclease digestion pattern, two recombinant plasmidswherein a DNA fragment of about 1.3 kb was inserted in the Bam HI siteof pUC8 were selected and designated as pBPDH1-DBL and pBPDH1-DBR,respectively. These plasmids are different in the orientation of the DNAfragment inserted therein.

E. coli transformants containing the plasmid thus obtained exhibitphenylalanine dehydrogenase activity.

Escherichia coli JM109/pBPDH1-DBL containing the plasmid pBPDH1-DBL wasdeposited with the FRI as FERM P-8873 on July 24, 1986; and Escherichiacoli JM109/pBPDH1-DBR containing the plasmid pBPDH1-DBR was depositedwith the FRI as FERM P-8794 on June 3, 1986.

Example 10. Production of phenylalanine dehydrogenase using transformantcontaining recombinant plasmid (1)

E. coli JM103/pBPDH1containing the plasmid pBPDH1, E. coli RR1/pBPDH3containing the plasmid pBPDH3, and E. coli JM103/pBPDH1-DBL containingthe plasmid pBPDH1-DBL were tested to determine their ability to producephenylalanine dehydrogenase.

The above-mentioned transformant E. coli were cultured in 140 ml of LBmedium containing 50 μg/ml of ampicillin at 37° C. for 6 hours. As acontrol, Bacillus sphaericus SCRC-R79a was cultured in 140 ml of amedium containing 0.1% of L-phenylalanine, 1% of peptone, 0.5% of yeastextract, 0.2% of K₂ HPO₄, 0.1% of NaCl, and 0.02% of MgSO₄ ·7H₂ O (pH7.0) at 37° C. overnight.

For each culture, cultured cells were collected by centrifugation, andthe cells were washed with 70 ml of a 0.85% NaCl solution. After thecells were suspended in 10 ml of 0.1 M potassium phosphate buffer (pH7.0), the suspension was ultra-sonicated at 2° C., 200 W, for 20minutes. The sonicate was dialyzed in 10 l of a 0.01 M potassiumphosphate buffer (pH 7.0) at 4° C. overnight. The dialyzate was assayedto determine the phenylalanine dehydrogenase activity according to theabove-mentioned procedure. The results are set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                                          Enzyme activity                                             Producer          (units/1 l culture)                                         ______________________________________                                        B. sphaericus SCRC-R79a                                                                         about       60                                              E. coli JM103/pBPDH1         1500                                             E. coli RR1/pBPDH3           2600                                             E. coli JH103/pBPDH1-DBL     3000                                             ______________________________________                                    

As seen from Table 1, E. coli transformed with a plasmid of the presentinvention produced 25 to 50 times as much phenylalanine dehydrogenasecompared with that produced by B. sphaericus.

Example 11. Synthesis of L-phenylalanine (1)

L-phenylalanine was synthesized using cells of E. coli RR1 containingthe plasmid pBPDH3. As an NADH regeneration system, enzymes forglycolysis in E. coli were used. The above-mentioned E. coli wasinoculated in an LB medium containing 50 μg/ml of ampicillin at a ratioof 1%, and cultured at 37° C. for 6 hours. The 5 ml of the culturedbroth was centrifuged to collect cells, which were then washed with a0.85% NaCl solution. The cells were suspended in 3 ml of a reactionmixture containing 200 μ moles of an NH₄ Cl-NH₄ OH buffer (pH 9.0), 109μ moles of a sodium phenylpyruvate, and 267 μ moles of a lactose, andthe suspension was allowed to stand at 30° C. for 24 hours. As a result,20 mg/ml (yield 33%) of L-phenylalanine was accumulated, as determinedby the above-mentioned L-phenylalanine assay procedure.

Example 12. Synthesis of L-phenylalanine (2)

In this example, as an NADH regeneration system, cells of Candidaboidinii No. 2201 containing formate dehydrogenase were used. Candidaboidinii No. 2201 was cultured according to the Tani method (literature2), and cells of C. boidinii and cells of E. coli RR1/pBPDH3 containingthe plasmid pBPDH3 cultured as above were separately treated withacetone (literature 3). Then, 15 mg of the acetone-treated cells of E.coli corresponding to 6.3 ml of the cultured broth having 6.3 units ofphenylalanine dehydrogenase activity, and 30 mg of the acetone-treatedC. boidinii cells were suspended in ml of a reaction mixture containing250 μ moles of a Tris-HCl buffer (pH 8.2), 728 μ moles (0.136 g) of aphenylpyruvic acid, 2 m moles (0.122 g) of an ammonium formate, and 25 μmoles (1.8 g) of NAD⁺, and the suspension was allowed to stand for 30°C. for 53 hours to synthesize L-phenylalanine. As a result, 0.126 g (42mg/ml) of L-phenylalanine was obtained. This yield corresponds to a 100%conversion.

Example 13. Preparation of chromosomal DNA (2)

According to the method of Doi et al. (literature 1), chromosomal DNAwas prepared from Sporosarcina ureae SCRC-R04 (FERM BP-1012).

Then, 1 l of a medium containing 1% of L-phenylalanine, 0.5% of yeastextract, 1.0% of peptone, 0.2% of KH , 0.1% of NaCl, and 0.02% of MgSO₄·7H₂ O was autoclaved, and the above-mentioned microorganism wasinoculated into the medium and cultured at 30° C. overnight.

The cultured broth was centrifuged to collect cells, which were thensuspended in 20 ml of a TEN buffer. The suspension was again centrifugedto collect the cells, which were then resuspended. To the suspension wasadded 1 ml of 5 mg/ml lysozyme, and the whole was shaken for 30 minutesat 37° C. The mixture was centrifuged to obtain a precipitate, which wasthen suspended in 5 ml of a TEN buffer. To the suspension were added 0.5ml of a 25% sodium dodecyl sulfate, 1 ml of a 5 M NaCl and 10 ml ofphenol saturated with water to extract DNA. The mixture was centrifugedto obtain a supernatant, and the supernatant was washed withchloroform/isoamylalcohol (24:1, v/v). The whole was then centrifuged,and the supernatant was slowly added to 50 ml of ethanol to precipitateDNA, which was then recovered. After drying the DNA, the DNA wasdissolved in a TEN buffer.

Example 14. Preparation of chromosomal DNA library (2)

The 50 μg of chromosomal DNA prepared in Example 13 was digested with 20units of a restriction endonuclease Eco RI at 37° C. for 2 hours, thereaction mixture was extracted with phenol/chloroform (1:1 v/v), and 2.5volumes of ethanol was added to the mixture to precipitate DNA.

On the other hand, 5 μg of the vector plasmid pUC9 was digested with 10units of the above-mentioned corresponding restriction endonuclease at37° C. for 2 hours. DNA was recovered as described above.

Then, 0.5 μg of the digested vector plasmid pUC9 and 4 μg of thechromosomal DNA fragment was ligated using a T4 DNA ligase to formrecombinant plasmids. The plasmids were then used to transform E. coliK-12/JM103 to construct a chromosomal DNA library. Each librarycomprised 20,000 to 30,000 transformants.

Example 15. Cloning of phenylalanine dehydrogenase gene (2)

The above-mentioned library was plated on nitrocellulose filters (0.45μm, TYPE TM-1, Toyo Roshi, Japan) put on an LB agar Plate containing 50μg/ml of ampicillin at a ratio of 1,000 to 2,000 colonies per filter,and incubated at 37° C. for 8 hours. Each filter was replicated to twonitrocellulose filters, and culturing was continued. The latternitrocellulose filter was soaked in 0.5 ml of 4 mg/ml of a lysozymesolution at a room temperature, and then dried at 45° C. for one hour.The filter was then soaked in 0.5 ml of an active staining solutioncontaining 50 mM of L-phenylalanine, 50 mM of Tris-HCl (pH 8), 0.625 mMof NAD⁺, 0.064 mM of phenazine methosulfate (PMS), and 0.24 M ofnitroblue tetrazalium (INT) at a room temperature for 1 to 5 minutes toselect reddish brown-stained colonies (literature 5). Some of theselected colonies were separately cultured in an LB medium containing 50μg/ml of ampicillin at 37° C. overnight, and the cultured cells weresonicated. The sonicate was then centrifuged to obtain a cell-freeextract, and to the extract was added the above-mentioned activestaining solution to detect enzyme activity. As a result, a transformantE. coli JM103/pSPDH1 was obtained. A plasmid pSPDH1 contained thephenylalanine dehydrogenase gene.

Example 16. Preparation of restriction endonuclease map and subcloning(2)

The transformant E. coli JM103/pSPDH1 was cultured in 200 ml of an LBmedium containing 50 μg/ml of ampicillin at 37° C. overnight, andaccording to method of Birnboim and Doly (literature 6), 150 μg ofplasmid DNA was obtained. The plasmid pSPDH1 DNA thus obtained wasdigested with a restriction endonuclease Bam HI, Eco RI, Bgl II, Pvu II,Nae I, Hinc II or the like to prepare a restriction endonuclease map(FIG. 4A).

An insert DNA of about 5.6 kb in pSPDH1 was fragmented to prepare a NaeI - Hind III DNA fragment of about 1.4 kb, the DNA was inserted at theHind III - Sma I site of the vector plasmid pUC19, and the resultingrecombinant plasmids were used to transform E. coli K-12/JM103 to obtaintransformant E. coli JM103/pSPDH2 capable of producing phenylalaninedehydrogenase. The restriction endonuclease map of pSPDH2 is set forthin FIG. 4-B.

The nucleotide sequence of the DNA fragment containing a region codingfor phenylalanine dehydrogenase in plasmids pSPDH1 and pSPDH2, as wellas an amino acid sequence corresponding to the nucleotide sequence, areset forth in FIGS. 5-1 to 5-3. The nucleotide sequence was determined bya dideoxy method (literature 4) and MB dideoxy chain termination method(literature 7).

The transformant Escherichia coli JM103/pSPDH2 was deposited with theFRI as FERM P-8793 in June 3, 1986.

Example 17. Production of phenylalanine dehydrogenase (2)

E. coli JM103/pSPDH2 and JM103/pSPDH1 were cultured in an LB mediumcontaining 50 μg/ml of ampicillin at 37° C. for 6 hours. On the otherhand, as a control, Sporosarcina ureae SCRC-R04 was cultured in themedium described in Example 1 at 30° C. for 16 hours. Each culturedbroth was centrifuged to collect cells, which were then suspended in a0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 5mM of 2-mercaptoethanol, and sonicated. The sonicate was centrifuged,and the supernatant was dialyzed in a 10 mM potassium phosphate buffer(pH 7.0) containing 0.1 M of EDTA and 5 mM of 2-mercaptoethanol at 4° C.overnight. These preparations were assayed according to theabove-mentioned method, and the results are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                                         Enzyme activity                                              Producer         units/1 l culture                                            ______________________________________                                        S. ureae CSRC-R04                                                                               60                                                          E. coli JM103/pSPDH1                                                                           175                                                          E. coli JM103/pSPDH2                                                                           340                                                          ______________________________________                                    

As seen from Table 2, recombinant E. coli of the present inventionproduced 6 times as much phenylalanine dehydrogenase compared to thatproduced by a wild strain of S. ureae.

Example 18. Synthesis of L-phenylalanine (3)

E. coli JM103/pSPDH2 was cultured according to the same procedure asdescribed in Example 17. On the other hand, Candida boidinii No. 2201was cultured according to the Tani method (literature 2) to prepare anNADH regeneration system. Each cultured broth was centrifuged to collectcells, which were then treated with acetone (literature 3) to prepare0.48 g/l of acetone-treated dry cells and 1.3 g/l of acetone-treated drycells from the E. coli and C. boidinii respectively.

Then, 3 ml of a reaction mixture containing 0.136 g (728 μ moles) ofsodium phenylpyruvate, 0.122 g (2 m moles) of ammonium formate, 1.8 mg(2.5 μ moles) of NAD⁺, 250 m moles of Tris-HCl (pH 8.5), 15 mg of the[acetone-treated E. coli cells (corresponding to 30 ml of culture, 6units of phenylalanine dehydrogenase), and 3 mg of the acetone-treatedC. boidinii cells (corresponding to 2.3 ml of culture, 0.14 units offormate dehydrogenase) was incubated at 30° C. for 53 hours.Accordingly, 0.120 g (yield 100%) of L-phenylalanine was obtained.

Example 19. Preparation of chromosomal DNA (3)

A chromosomal DNA was prepared from Bacillus badius IAM 11059 (FERMP-8529) according to the method of Doi et al (literature 1).

That is, the above-mentioned strain was cultured in 1.2 l of a mediumcontaining 1% of L-phenylalanine, 0.5% of yeast extract, 1.0% ofpeptone, 0.2% of K₂ HPO₄, and 0.02% of MgSO₄ ·H₂ O (pH 7.0) at 30° C.for 6 hours. Extraction of the chromosomal DNA was carried out accordingto the same procedure as described in Example 1.

Example 20. Preparation of chromosomal DNA library (3)

Next, 50 μg of the chromosomal DNA prepared in Example 19 was digestedwith 300 units of restriction endonuclease Eco RI at 37° C. overnight,the digest was extracted with phenol/chloroform (1:1, v/v), and DNA wasprecipitated with 2 volumes of ethanol. On the other hand, 10 μg of thevector plasmid pUC19 was digested with 60 units of Eco RI at 37° C.overnight, and DNA was recovered in the same manner as described above.

Then, 0.5 μg of the Eco RI-digested vector plasmid pUCI9 and 1 μg of theEco RI-digested chromosomal DNA were ligated using DNA ligase, and thereaction mixture was used to transform E. coli K-12/JM103 and prepare achromosomal DNA library. This chromosomal DNA library comprised 23,000transformants.

Example 21. Cloning of phenylalanine dehydrogenase gene (3)

The above-mentioned transformants were plated on nitrocellulose filtersput on an LB agar plate containing 50 μg/ml of ampicillin at a ratio of1000 to 2000 colonies per nitrocellulose filter, and then cultured.After about 10 hours, colonies on the nitrocellulose were replicatedonto fresh nitrocellulose filters, and culturing was continued. Thelatter nitrocellulose filter was soaked in 1.0 ml of a 4 mg/ml lysozymeaqueous solution at 30° C. for 10 minutes, followed by 10 ml of a 0.1 MTris-HCl (pH 8.0) at 50° C. for 10 minutes. The nitrocellulose filterswere soaked in 1.0 ml of an active staining solution containing 50 mM ofTris-HCl (pH 8.0), 0.625 mM of NAD⁺, 0.065 mM of PMS, and 0.24 mM of INTat a room temperature for 1 to 5 minutes, and the colonies stained to areddish violet color were selected (literature 5). From about 23,000transformants was obtained about 50 transformants exhibiting anactivity. Among these transformants, 12 transformants were separatelycultured in 1 l of an LB medium containing 50 μg/ml ampicillin, andaccording to the method of Birnboim and Doly (literature 6) a plasmidDNA was prepared from each culture. These plasmid DNA's were analyzed todetermine the gene size thereof, using agarose gel. As a result, it wasdetermined that one clone had a gene fragment of about 7 kb insertedtherein, and 11 clones had a gene fragment of about 3.8 kb insertedtherein. All of these transformants exhibited phenylalaninedehydrogenase activity. Among these transformants, a transformantcontaining a plasmid having the 3.8 kb gene fragment was designated asE. coli JM103/pBBPDH19.

Example 22. Preparation of restriction endonuclease map (3)

The transformant E. coli JM101/pBBPDH19 was cultured in 1 l of an LBmedium containing 50 μg/ml of ampicillin at 37° C. overnight, andaccording to the method of Birnboim and Doly (literature 6), about 150μg of a plasmid DNA was prepared. The plasmid pBBPDH19 DNA wasseparately digested with a restriction endonuclease Sma I, Xma I, BamHI, Eco RI, Hind III, Pst I, Sal I, Acc I, and the like, to prepare arestriction endonuclease map (FIG. 6).

The transformant E. coli JM103/pBBPDH19 was cultured and the plasmidpBBPDH19 was isolated as above. This plasmid was used to transform E.coli RR1 to obtain a transformant Escherichia coli RR1/pBBPDH19, whichwas deposited with the FRI as FERM P-8890 on Aug. 7, 1986.

Example 23. Production of phenylalanine dehydrogenase (3)

E. coli RR1/pBBPDH19 was cultured in an LB medium containing 50 μg/mlampicillin at 37° C. for 12 hours. On the other hand, Bacillus badiusIAM 11059 (FERM P-8529) as a control was cultured in the mediumdescribed in Example 1 at 30° C. for 20 hours. Each cultured broth wascentrifuged to collect cells, which were then suspended in 0.1 M of apotassium phosphate buffer containing 0.1 mM EDTA and 5 mM2-mercaptoethanol, sonicated, the sonicate was centrifuged, and thesupernatant was dialyzed in a 10 mM potassium phosphate buffercontaining 0.1 mM of EDTA and 5 mM of 2-mercaptoethanol, overnight.These cell-free extracts were assayed to determine the phenylalaninedehydrogenase activity thereof, according to the above-mentionedprocedure. The results are set forth in Table 3.

                  TABLE 3                                                         ______________________________________                                                         Enzyme activity                                              Producer         (units/1 l culture)                                          ______________________________________                                        B. badius IAM 11059                                                                            1,090                                                        E. coli RR1/pBBPDH19                                                                           6,900                                                        ______________________________________                                    

As seen from Table 3, E. coli containing the recombinant plasmid of thepresent invention exhibited an phenylalanine dehydrogenase activity thatwas about 6 to 7 times greater than that of a wild Bacillus badiumstrain.

Example 24. Purification of phenylalanine dehydrogenase from E. coliRR1/pBBPDH19

E. coli RR1/pBBPDH19 was cultured in 1.8 l of an LB medium containing 50μg/ml of ampicillin at 37° C. for 12 hours. The cultured broth wascentrifuged to collect cells, which were then washed with aphysiological saline, and suspended in 1 l of a 0.1 M phosphate buffer(pH 7.0) containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. Thesuspension was sonicated at 9 kHz for one hour to disrupt the cells, andthe sonicate was centrifuged at 14,000 xg for 20 minutes to eliminatecell debris, so that a crude cell-free extract containing phenylalaninedehydrogenase was obtained. After the cell-free extract was heated at50° C. for 10 minutes, a solid ammonium sulfate was added to thecell-free extract to a concentration of 30% ammonium sulfate saturation.The whole was stirred for 30 minutes to allow the formation of aprecipitate, and centrifuged at 14,000 xg for 20 minutes to eliminatethe precipitate. Solid ammonium sulfate was added to the supernatant toa 60% ammonium sulfate saturation. The whole was the centrifuged at14,000 xg for 20 minutes to recover a precipitate containing enzymeactivity, and the precipitate was dissolved in a small volume of a 0.01M phosphate buffer (pH 7.0), and the resultant solution was dialyzed ina 0.01 M phosphate buffer (pH 7.0) containing 0.1 mM of EDTA and 5 mM of2-mercaptoethanol. This enzyme solution was applied to a DEAE-Toyopearl650 M column previously equilibrated with a 0.01 M phosphate buffer (pH7.0) containing 0.1 mM of EDTA and 5 mM of 2-mercaptoethanol, and theelution was carried out using a 0.1 M phosphate buffer (pH 7.0)containing 0.1 mM of EDTA and 5 mM of 2-mercaptoethanol.

Active fractions were then combined and dialyzed, and the dialyzate wasconcentrated and subjected to gel filtration chromatography usingSephadex G-200 equilibrated with a 0.05 M phosphate buffer (pH 7.0)containing 0.1 mM of EDTA and 5 mM of 2-mercaptoethanol. By thisprocedure, phenylalanine dehydrogenase was purified to an about 15-folddegree with a yield of about 54%. The specific activity and recoverypercentage during this purification procedure are set forth in Table 4.It was confirmed that the final enzyme preparation was homogeneous, asshown by polyacrylamide gel electrophoresis and DS-polyacrylamide gelelectrophoresis.

                  TABLE 4                                                         ______________________________________                                                      Total   Total   Specific                                                                              Recov-                                                activity                                                                              protein activity                                                                              ery                                     Step          (units) (mg)    (units/mg)                                                                            (%)                                     ______________________________________                                        1.  Cell-free extract                                                                           12,400  1,760 7.05    100                                   2.  Heat treatment                                                                              11,900  1,310 8.78    96                                    3.  Ammonium sulfate                                                                            7,680   942   7.91    62                                        fraction                                                                  4.  DEAE-Toyopearl                                                                              7,100   299   23.7    57                                    5.  Sephadex G-200                                                                              6,710     98.8                                                                              67.9    54                                    ______________________________________                                    

Example 25. Synthesis of L-phenylalanine (4)

E. coli RR1/pBBPDH19 was cultured as described above. On the other hand,Candida boidinii No. 2201 was cultured according to the method of Taniet al. (literature 2) to prepare an NADH regeneration system. Eachcultured broth was centrifuged to collect cells, which were then treatedwith acetone to obtain a 0.5 g/l culture of cetone-treated E. coli cellsand a 1.3 g/l culture of acetone-treated C. boidinii cells.

Then, 3 ml of a reaction mixture containing 0.136 g (728 μ moles) ofsodium phenylpyruvate, 0.122 g (2 m. moles) of ammonium formate, 1.8 mg(2.5 μ moles) of NAD⁺, 250 μ moles of Tris-HCl (pH 8.5), 5 mg ofacetone-treated E. coli cells (corresponding to 10 ml of the culture, 69units of L-phenylalanine dehydrogenase), and 3 mg of the acetone-treatedC. boidinii cells (corresponding to 2.3 ml of the culture, 0.14 units offormate dehydrogenase) was incubated at 30° C. for 48 hours.Subsequently, 0.120 g (yield 100%) of L-phenylalanine was obtained.

For the following microoganisms the national depositions were transferedto the international depository under the Budapest treaty on Aug. 11,1987:

    ______________________________________                                                        National    International                                     strain          deposition. No.                                                                           deposition No.                                    ______________________________________                                        E. coli J103/pSPDH2                                                                           FERM P-8793 FERM BP-1435                                      E. coli J109/pBPDH1-DBR                                                                       FERM P-8794 FERM BP-1434                                      E. coli J109/pBPDH1-DBL                                                                       FERM P-8873 FERM BP-1433                                      E. coli RR1/pBBPDH19                                                                          FERM P-8890 FERM BP-1436                                      ______________________________________                                    

Literature

(1) R. L. Rodriguez et al. Recombinant DNA Teqhnique, An Introduction,Addison-Wesley Publishing Company (1983) pp 162.

(2) Y. Tani et al. Agric. Biol. Chem., 36, 68, (1972).

(3) Y. Izumi et al. J. Ferment. Technol., 61, 135 (1983).

(4) H. Hattori et al. Nucl. Acids Res., 13, 7813 (1985).

(5) H. Mollering et al. Methods of Enzymatic Analysis Academic Press, 1,136-144 (1974).

(6) H. C. Birnboim et al. Nucl. Acids Res., 7, 1513 (1979).

(7 F. Sanger et al. Proc. Natl. Acad. Sci. USA 20 5463 (1977).

We claim:
 1. An isolated gene coding for phenylalanine dehydrogenase ofSporosarcina urea SCRC-R04 (FERM BP-1012).
 2. An isolated gene accordingto claim 1, wherein the gene comprises the following nucleotidesequence: ##STR1##
 3. An expression plasmid containing the gene ofclaim
 1. 4. An expression plasmid according to claim 3 wherein theplasmid is pSPDH2.
 5. A microorganism transformed with the plasmid ofclaim
 3. 6. A microorganism according to claim 5, wherein themicroorganism is E. coli.
 7. A microorganism according to claim 6,wherein the E. coli is E. coli JM103/pSPDH2 (FERM P-8793).
 8. A processfor production of phenylalanine dehydrogenase comprising the stepsof:culturing the microorganism of claim 5, and recovering thephenylalanine dehydrogenase from the cultured product.